1. Field of Invention
The present invention relates to side entry specimen cryotransfer holders for transmission electron microscopy and particularly to cryoholders capable of rotating the specimen in the plane of the specimen and tilting that specimen around the electron beam axis.
2. Description of Prior Art
Electron microscope specimen holders may either take the form of a cartridge or a rod. Cartridge type holders are inserted vertically into the microscope through the upper polepiece of the objective lens, whereas rod type holders are inserted horizontally through the side of the microscope between the upper and lower polepieces. In recent years, rod type holders have become more widely used because mechanisms for tilting the specimen are simpler and more reliable. The ability to tilt a specimen is important for stereo imaging, three dimensional reconstruction, and tuning for optimum diffraction conditions.
Rod type specimen holders have also become the preferred type for observation of specimens at low temperatures. This is because the extraction of heat from the specimen is easily accomplished by forming the specimen tip of a thermally conductive material such as copper and extending the tip in the form of a copper rod to a dewar of liquid nitrogen or helium mounted at the other end of the rod. Cryotransfer holders are a more recent development of the rod type cooling holders in which specimens can be transferred at low temperature into the microscope from an external cryostation without frosting of the specimen.
Conventional resin embedding methods for biological samples have been shown to produce readily identifiable artifacts that can be interpreted in reference to structures observed in living cells. Water must be removed from specimens during resin embedment, causing diffusion artifacts and collapse of delicate structures. For these and other reasons, many laboratories use ultra-rapid freezing technologies to prepare biological specimens for examination in the transmission electron microscope. While this approach has many advantages, several technical problems must be surmounted; the specimen must be rapidly frozen to promote the formation of amorphous ice, and the sample must be maintained at temperatures below xe2x88x92140xc2x0 C. to prevent devitrification.
Cryotransfer holders have been designed to maintain specimens in a frozen state and to prevent frost deposition on specimens during the transfer of the holder from a workstation into the microscope. Typically, when the specimen has been clamped into the holder, a cryo-shutter is moved to completely cover the specimen while being transferred. An example of this type of cryotransfer holder is described in Swann et al, U.S. Pat. No. 4,703,181. Once inserted into the microscope, the shutter is retracted to expose the frozen specimen. Depending on the type of specimen examined, the holder can require tilting to present the most advantageous aspect for recording. The cryoshutter can be replaced over the specimen if the specimen is destined for reexamination at a later date.
As the resolving power of electron microscopes has improved, efforts to resolve high resolution structures of biological organelles and macromolecules have been attempted. Three-dimensional electron microscopic imaging, or electron tomography, is one of the methods employed to gather detailed volume and surface data. This technique involves the reconstruction of individual objects from projection data collected over a large range of specimen tilts. An example of a cryotransfer holder which is useful in electron tomography is described in Swann, U.S. Pat. No. 5,753,924. A low resolution data set would include images of a specimen through a single tilt range of xc2x170xc2x0 at 1xc2x0 intervals. Ideally an object should be tilted xc2x190xc2x0, but current microscope design does not permit this range of movement.
Thus, practical limitations of tomography are set by the damage a specimen suffers with repeated irradiation and the range of specimen tilt available to generate projection data. To address the problem of specimen damage from repeated irradiation, samples are examined at low temperature in a cryoholder. The tilt range of a specimen could be extended by tilting the specimen in a second axis perpendicular to the first as is done with a double tilt holder. An example of a commercially-available double tilt holder is the Model 915 Double Tilt Cryotransfer System from Gatan, Inc., Pleasanton, Calif. For most double tilt holders, the tilt range of the second axis is limited to up to xc2x145xc2x0 by the construction of the specimen cradle or the method by which tilting through the second axis is achieved. A data set including xc2x170xc2x0 data in the first axis and xc2x145xc2x0 in the second axis of tilt is not ideal. The specimen cannot be removed from the microscope and manually reoriented after one set of tilt data is taken because there is a high probability that the specimen will melt and become damaged or contaminated during this manipulation.
As discussed above, if a conventional single tilt cryotransfer holder is used to collect a tomographic series, current data sets typically cover a range up to xc2x170xc2x0 of tilt through a single axis. When used for applications requiring high tilt such as tomography, this limited tilt range prohibits obtaining the necessary images for three-dimensional reconstruction of a specimen""s top and bottom surfaces. This comprises roughly one-third of the data set for the structure being examined, while ideally for a single tilt axis stage, a complete data set from xc2x190xc2x0 is needed to minimize directional distortions. However, this is impossible with currently-available cryotransfer holders.
In a known cryotransfer holder, designed and manufactured by Gatan Inc., a frozen specimen on a standard circular 3 mm diameter grid may be transported from a cryostation to the transmission microscope for examination. The cryo-holder is designed to maintain a specimen at a temperature of less than xe2x88x92160xc2x0 C. at all times. The specimen tip is configured to produce a primary axis tilt of xc2x170xc2x0 in a microscope polepiece gap with a xe2x80x9cZxe2x80x9d axis distance of 3 mm or larger from the centerline of the holder to the nearest contacting surface. The tip of the specimen rod contains a moveable cryo-shutter which prevents frost formation on the specimen during transfer from a workstation to the microscope. The cryoshutter is manipulated at will by means of a manual control positioned outside of the holder. The specimen grid is held in place by a specimen clamping device, for example, a Clipring (trademark of Gatan, Inc.).
Accordingly, the need still exists in this art for a cryotransfer holder which is capable of acquiring tilt data at high angles at more than one specimen orientation without disturbing the frozen specimen.
The present invention meets that need by providing a cryotransfer holder which takes advantage of the design features of the standard cryotransfer holders, including a moveable cryoshutter, maintaining a specimen temperature of less than xe2x88x92160xc2x0 C., and secure specimen clamping, but which also is capable of acquiring tilt data at high angles at more than one specimen orientation without disturbing the frozen specimen (i.e., without removing the specimen from the microscope). The present invention adds the ability to rotate the specimen in the plane of the specimen at cryotemperatures during observation in the microscope which extends the useful maximum tilt range of the holder through another tilt axis, ideally perpendicular to the original axis.
In accordance with one aspect of the present invention, a side-entry specimen cryoholder for an electron microscope is provided and includes a specimen holder including a specimen cradle having a specimen grid adapted to carry a specimen to be analyzed; a translation mechanism for rotating the specimen cradle within the specimen holder in the plane of the specimen; a mechanism for tilting the specimen cradle; and a cryoshutter which is adapted to protect the specimen during cryotransfer and moveable from a first position covering the specimen to a retracted position. Preferably, the translation mechanism includes an actuation mechanism which is operable externally of the electron microscope. Also preferably, the specimen cryoholder includes a source of cooling.
In a preferred embodiment of the invention, the translation mechanism comprises a motion exchange mechanism linked to the specimen cradle and a translation shaft linked to the motion exchange mechanism. The translation shaft preferably comprises a rod having first and second ends and extending along the long axis of the specimen holder, with the first end of the rod being connected to the motion exchange mechanism and the second end adapted to extend outside the transmission electron microscope. In one embodiment, the motion exchange mechanism comprises a toothed rack communicating with corresponding toothed gears on the periphery of the specimen cradle. The cryoholder further includes a knob threadably connected to the specimen holder, with the second end of the translation shaft being coupled linearly to the knob such that rotation of the knob causes linear movement of the translation shaft.
A sliding vacuum seal communicating with the translation shaft is also provided such that the vacuum inside the transmission electron microscope is maintained during movement of the motion exchange mechanism. The cryoshutter also includes a pin connected therewith such that linear movement of the translation mechanism engages the pin and causes the cryoshutter to move from the first position covering the specimen to a retracted position. The source of cooling preferably comprises a dewar of liquid nitrogen connected to the specimen holder by a thermally insulated conductor.
In operation, the specimen cryoholder is inserted into an electron microscope and the translation mechanism is actuated to retract the cryoshutter. A first set of data is taken with the specimen in a first orientation and the specimen cradle tilted through approximately xc2x170xc2x0. Then, the specimen is rotated, preferably 90xc2x0 in the plane of the specimen using the motion exchange mechanism, and a second set of tilt data is taken through approximately xc2x170xc2x0. This procedure adds a second set of tilt data to compensate for information which would otherwise be missing from prior art specimen holders unless the holder were removed from the microscope and the specimen manually rotated. The present invention facilitates the production of a high resolution reconstruction of the object being studied without further handling of the frozen specimen.
Accordingly, it is a feature of the invention to provide a side-entry cryotransfer holder for electron microscopes that allows a specimen to be rotated in a plane perpendicular to the electron beam axis in a transmission electron microscope without removing the specimen from the microscope. It""s a further feature of the invention to provide a rotation mechanism which is activated externally of the microscope. It is yet a further feature of the invention to provide a moveable cryoshutter which protects the specimen during transfer from the specimen loading workstation into a microscope, which also protect regions of the grid when the holder is positioned in the microscope before viewing of the sample, and which is removed prior to a viewing session. These, and other features and advantages of the present invention, will become apparent from the following detailed description, the accompanying drawings, and the appended claims.